Through its role in the maintenance of the Na,K-electrochemical gradient, the sodium, potassium-adenosine triphosphatase (Na,K-ATPase) is involved in nerve conduction, contractility, myocardial, inotropy, vascular resistance, renal ion-solute uptake. It is not surprising, therefore, to find Na<K- ATPase alphasubunit isoform modulation secondary to hypertension as well as possible involvement in the polygenic etiology of genetic hypertension. The dissection of the molecular mechanisms of its primary and secondary roles in hypertension is imperative. This proposal therefore aims to: (I) identify the mechanisms of modulation, and when transcriptional, the cis- acting regulatory elements and factors involved in the regulation of Na,K- ATPase alpha1 and alpha2 gene expression in hypertension; and (II) screen human hypertensive patients for mutations in the Na,K-ATPase alpha1 gene parallel to the mutations found in the Na,K-ATPase alpha1 gene of the genetic hypertensive rat model, Dahl-salt sensitive rat. Briefly, the specific aims and corresponding methods are as follows: 1) to complete the characterization of the 5' flanking regions of alpha1 and alpha2 genes by the identification of the respective transcription initiation sites and consensus cis-acting regulatory sequences; 2) to delimit the putative functional 5' flanking region of alpha1 and alpha2 genes, as marked by the presence of tissue specific cis-acting regulatory regions in a tissue culture system using chimeric gene constructs with the chloramphenicol acetyl transferase gene (CAT) as reporter function gene; 3) to assess whether transcriptional and/or post-transcriptional mechanism(s) are involved in the modulation of alpha1 and alpha2 expression in hypertension using transgenic mice; 4) to begin to identify transacting regulatory proteins that recognize putative hypertensive regulatory elements, and their respective cDNAs by southwestern blotting techniques and southwestern cDNA screening of hypertensive heart-aorta cDNA expression libraries; and 5) to begin to identify subsets of hypertensive patients with one or more mutations in the Na,K-ATPase alpha1 gene by using DNA amplification techniques and strategic oligonucleotide screening. The information to be obtained from these studies will be critical towards ushering in approaches to study "regulatory master genes" centrally involved in hypertension, as well as, hopefully, a genetic marker for a subset of human hypertensive patients.